阅读说明：本技术 萝卜硫素在提高哺乳动物繁殖性能中的应用 (Application of sulforaphane in improving reproductive performance of mammals ) 是由 刘红林 陶景丽 许波 张轩 周佳奇 张良良 李伟建 李荣阳 于 2020-07-20 设计创作，主要内容包括：萝卜硫素在提高哺乳动物繁殖性能中的应用,本发明利用萝卜硫素能够促进体外颗粒细胞的增殖和活力、抑制颗粒细胞的凋亡。萝卜硫素体外促进雌二醇的生成以及颗粒细胞StAR、CYP19A1等类固醇合成酶基因的表达。萝卜硫素促进体外颗粒细胞FSH受体的表达。萝卜硫素促进小鼠卵巢发育,增加卵巢重量。萝卜硫素促进体内颗粒细胞的增殖。萝卜硫素促进小鼠卵巢卵泡发育,增加卵泡数,增加排卵数。断奶母猪日粮添加萝卜硫素显著提高了母猪产仔数。本发明应用萝卜硫素提高哺乳动物繁殖性能的技术,提供了萝卜硫素的新用途,极大的提高了其应用价值,扩大了萝卜硫素的应用范围,拓展出应用萝卜硫素的新领域,具有广阔的应用前景和商业价值。(The invention relates to an application of sulforaphane in improving the reproductive performance of mammals, which can promote the proliferation and the activity of in vitro granular cells and inhibit the apoptosis of the granular cells by utilizing the sulforaphane. Sulforaphane promotes the production of estradiol and the expression of steroid synthase genes such as granulosa cells StAR and CYP19A1 in vitro. Sulforaphane promotes the expression of the FSH receptor of granulosa cells in vitro. Sulforaphane promotes development of mouse ovary and increases weight of ovary. Sulforaphane promotes the proliferation of granulosa cells in vivo. The sulforaphane promotes the development of ovarian follicles of mice, increases the number of the follicles and increases the number of ovulations. The daily ration of the weaned sow added with the sulforaphen obviously improves the litter size of the sow. The invention provides a new application of the sulforaphane, greatly improves the application value of the sulforaphane, enlarges the application range of the sulforaphane, expands the new field of the sulforaphane, and has wide application prospect and commercial value.)

1. Application of sulforaphane and its derivatives in preparing product for improving mammal reproductive performance is provided.

2. Application of sulforaphane and derivatives thereof in preparing mature products for promoting follicular granulosa cell proliferation.

3. Application of sulforaphane and derivatives thereof in preparing products for promoting ovarian follicle development.

4. Application of sulforaphane and derivatives thereof in preparing products for promoting estrus and ovulation.

5. The use according to claim 1, characterized in that the effective concentration of sulforaphane is: not higher than 10 μ M in vitro and not higher than 5mg/kg in vivo.

6. The use according to claim 5, characterized in that the effective concentration of sulforaphane is: 1-10 μ M in vitro and 0.1-5 mg/kg in vivo.

Technical Field

The invention belongs to the technical field of reproductive biology, and particularly relates to application of sulforaphane in improving reproductive performance of mammals.

Background

The reproduction is the root of the continuation of the human generation, unfortunately, the reproduction disorder of the human beings in the current society occurs frequently, and about 10 percent of couples are troubled by the reproduction disorder according to statistics; on the other hand, the reproductive performance of livestock is an important economic character, and the improvement of the double-lamb rate of livestock such as beef cattle, sheep and the like and the increase of the litter size of sows (shortening the gap with developed countries) have great significance for improving the quality and the efficiency of the animal husbandry. Therefore, the research and development of a new technology for safely and efficiently improving the reproductive performance of mammals not only has great practical requirements, but also has great application value and commercial prospect.

The ovary is the most important reproductive organ of female animals, and the function of the ovary is mainly to produce ovum and steroid hormone. Ovarian follicle growth and development are the physiological basis for oestrus and ovulation in dams. Granulosa cells are the main components of follicles except oocytes in follicles, and the regulation of the growth and development of oocytes by the production of steroid hormones (e.g., estradiol and progesterone) and the secretion of Stem Cell Factor (SCF) is an important function of granulosa cells (Yada H et al, 1999). During the development of the follicle, granulosa cells are continuously proliferated and differentiated, and are expressed as morphological change and increase of the number of cells and the number of layers; at the same time, the granulosa cells have an increasing capacity for steroid hormone synthesis. Follicular development is mainly regulated by Follicle Stimulating Hormone (FSH), which promotes cell proliferation and steroid hormone production via granulosa cell membrane receptors (Yu FQ et al, 2005). FSH is considered to be the most important regulator of follicular growth and development. It is known that exogenous administration of FSH is effective in promoting ovulation and that techniques for superovulation in various domestic animals including pigs have been established using this principle, but these techniques have the disadvantages of high cost, influence on hormone balance and embryo attachment, etc. The applicant finds that the plant-derived small molecular compound-Sulforaphane (SFN) can remarkably promote the proliferation of porcine follicular granular cells and mouse follicular granular cells, and surprisingly, the promoting effect of the SFN on the proliferation of the porcine follicular granular cells is better than that of FSH, and the expression of steroid synthase genes such as StAR, CYP19A1 (similar to FSH) is remarkably promoted, so that the possibility of improving the reproductive performance of female animals by utilizing the SFN is provided.

Sulforaphane (1-isothiocyanato-4-methylsulfonylbutane, SFN), also known as sulforaphane, is an isothiocyanate produced from glucosinolates by myrosinase hydrolysis in plants, and is widely found in cruciferous plants, such as broccoli, cabbage, cauliflower, and the like. Sulforaphane is a sulfur-containing compound with molecular formula of C₆H₁₁S₂NO, with a relative molecular mass of 177.29, is a yellow or colorless liquid at room temperature, is insoluble in water, is very soluble in organic solvents, and is easy to absorb. A large number of research reports prove that sulforaphane has the functions of resisting oxidation, tumors and inflammation, regulating epigenetic modification and the like (Li H et al, 2018; Byrne MM et al, 2014). It has been shown that ingestion of sulforaphane-rich cruciferous vegetables is beneficial for health, including prevention of many diseases such as cardiovascular diseases, neurodegenerative diseases and diabetes. In terms of tumor prevention, sulforaphane can reduce the risk of many types of tumors, such as colorectal cancer, renal cancer, breast cancer, and the like. Regulating and controlling cell proliferation and differentiation are important ways for SFN to play a role, the concentration and cell type of the SFN influence the regulating and controlling effect of the SFN on cell proliferation and differentiation, for example, Han et al (Han Z et al, 2017) find that low-concentration sulforaphane can promote the proliferation of neural stem cells and remarkably promote the differentiation of neurons, so that the effect of preventing neurodegenerative diseases is achieved; in contrast, sulforaphane has the effects of inhibiting the proliferation of different types of cancer cells, inducing cell cycle arrest of the cancer cells, promoting cancer cell apoptosis and the like (Bryant CS et al, 2010; Chaudhuri D et al, 2007; Ashok BT et al, 2005).

The present invention related to sulforaphane focuses on its extraction and preparation technology, such as a preparation method for extracting sulforaphane from broccoli (CN105949098A), a sulforaphane extraction process (CN108048498A), a sulforaphane extraction method (CN108912024A), a sulforaphane microcapsule preparation method (CN107569470A), a sulforaphane inclusion compound preparation method (CN108992675A), a sulforaphane health tablet preparation method (CN106214721A), and the like. In the application aspect of the sulforaphane, patents focusing on the protection effect of the sulforaphane on adult health are mostly focused, and no patents for promoting the reproductive performance of mammals exist, such as the application of the sulforaphane in telomerase activity inhibition and preparation of anti-cancer drugs (CN109771409A), the application of the sulforaphane in preparation of foods, health care products or medicines for preventing diabetic lesions (CN108743576A), the application of the sulforaphane in preparation of drugs for protecting the fetal vascular development of women drinking wine during pregnancy (CN108771669B) and the like.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides an application of sulforaphane in improving the reproductive performance of mammals in order to improve the reproductive capacity of the mammals. The sulforaphane is adopted to remarkably promote the proliferation of follicular granular cells of pigs and mice and the synthesis of steroid hormones, and more importantly, the sulforaphane with a certain concentration can promote the ovarian weight gain of the mice, the follicular development and ovulation of the mice and the litter size of sows.

The technical scheme is as follows: application of sulforaphane and its derivatives in preparing product for improving mammal reproductive performance is provided.

Application of sulforaphane and derivatives thereof in preparing mature products for promoting follicular granulosa cell proliferation.

Application of sulforaphane and derivatives thereof in preparing products for promoting ovarian follicle development.

Application of sulforaphane and derivatives thereof in preparing products for promoting estrus and ovulation.

The effective concentration of the sulforaphane is as follows: not higher than 10 μ M in vitro and not higher than 5mg/kg in vivo.

The effective concentration of the sulforaphane is as follows: 1-10 μ M in vitro and 0.1-5 mg/kg in vivo.

The Sulforaphane (SFN) has the molecular formula of C₆H₁₁S₂NO, relative molecular mass 177.29.

Has the advantages that: the sulforaphane promotes the proliferation and the activity of in vitro granular cells and inhibits the apoptosis of the granular cells. Sulforaphane promotes the production of estradiol and the expression of steroid synthase genes such as granulosa cells StAR and CYP19A1 in vitro. Sulforaphane promotes the expression of the FSH receptor of granulosa cells in vitro. Sulforaphane promotes development of mouse ovary and increases weight of ovary. Sulforaphane promotes the proliferation of granulosa cells in vivo. The sulforaphane promotes the development of ovarian follicles of mice, increases the number of the follicles and increases the number of ovulations. The daily ration of the weaned sow added with the sulforaphen obviously improves the litter size of the sow. The invention provides a new application of the sulforaphane, greatly improves the application value of the sulforaphane, enlarges the application range of the sulforaphane, expands the new field of the sulforaphane, and has wide application prospect and commercial value.

Drawings

FIG. 1 is a graph showing that sulforaphane promotes the proliferation and viability of granulosa cells in vitro and inhibits apoptosis of the granulosa cells in example 1 of the present invention. In the figure, A: after the in vitro culture granular cells of the pigs and the mice are treated by adding the sulforaphane for 24 hours, the activity level of the cells is detected by CCK-8. B: after the granular cells cultured in vitro of the pigs and the mice are added with the sulforaphane for treatment for 12h, the granular cells are doped with EdU for incubation for 2h, and then the granular cells are observed and photographed under a laser confocal microscope, the level of the EdU staining marker of the granular cells is detected, and the proportion of positive cells is quantitatively analyzed. C: after the granular cells cultured in vitro of pigs and mice are treated by adding sulforaphane for 12 hours, the expression level and the gray level of PCNA protein of the granular cells are detected by western blot. D: adding sulforaphane into the mouse in vitro cultured granular cells, and detecting the expression level and gray level analysis of the granular cell clean-caspase 3 protein by using western blot after 12 hours. The results of the above corresponding experiments are expressed as mean ± sem, # P <0.05, # P <0.01, # P <0.001, # P <0.0001, ns, not significant, P > 0.05.

FIG. 2 is a graph showing the in vitro promotion of estradiol production by sulforaphane and the expression of steroid synthase genes such as granulosa cells StAR and CYP19A1 in example 2 of the present invention. In the figure, A: ELISA was used to detect changes in estrogen levels in the supernatants of granular cell cultures from control and sulforaphane mice. P < 0.05. B: after the mouse granular cells are treated by the sulforaphane for 12 hours, the mRNA level change of the steroid hormone synthetase gene is detected by fluorescent quantitative PCR, and the expression quantity of the related gene is calculated by taking beta-actin as an internal reference. C: after the mouse granular cells are treated by sulforaphane for 12 hours, Western blot is used for detecting the protein level change of the steroid hormone synthetase gene and carrying out gray level analysis. The results of the above corresponding experiments are expressed as mean ± sem, # P <0.05, # P <0.01, # P <0.001, # P <0.0001, ns, not significant, P > 0.05.

FIG. 3 is a graph showing the enhancement of in vitro granulosa cell FSH receptor expression by sulforaphane in example 3 of the present invention. After the mouse granular cells are treated by sulforaphane for 12 hours, Western blot is used for detecting the level change of FSHR protein of an FSHR receptor and carrying out gray scale analysis. Results are expressed as standard deviation ± sem. P < 0.01.

FIG. 4 is a graph of sulforaphane promoting development of mouse ovary and increasing weight of ovary in example 4 of the present invention. In the figure, A: the development of ovaries is promoted after the sulforaphane with different concentrations is injected into abdominal cavities of mice (n is 7), so that the ovaries are enlarged. B: the influence and statistics of different concentrations of sulforaphane on the change of the weight of the ovary after the intraperitoneal injection (n is 7) of the mouse are changed. C: after intraperitoneal injection of mice (5 weeks old) with S6 concentration of sulforaphane according to the A, B results, mouse ovaries of the control group, sulforaphane and follitropin group were weighed and counted together, respectively (n ═ 18).

FIG. 5 is a graph showing that sulforaphane promotes the proliferation of granulosa cells in vivo in example 5 of the present invention. In the figure, A: after the mice are injected with the sulforaphen in the abdominal cavity, the microscopic confocal detection respectively detects the level of the ovarian granular cell Edu staining markers of the control group, the sulforaphen group and the follicle-stimulating hormone group and the quantitative statistics of the proportion of the marked cells. B: after injecting sulforaphen into the abdominal cavity of the mouse, detecting the change of the levels of the proliferation marker protein PCNA of the control group and the sulforaphen group by Western blot, and performing gray level analysis. The corresponding test results are expressed as mean ± sem. P <0.001, P <0.0001, ns, not significant, P > 0.05.

FIG. 6 is a graph showing that sulforaphane promotes the production of estradiol in vivo and that steroid synthase genes such as granulosa cells StAR and CYP19A1 are expressed in example 6 of the present invention. In the figure, A: radioimmunoassay was used to detect the levels of estrogen and progestin in the serum of control and sulforaphane mice. B: and (3) analyzing the expression level and the quantification of the ovarian steroid hormone synthetase protein of the control group and the sulforaphane group mice. The results of the above corresponding experiments are expressed as mean ± sem, # P <0.05, # P <0.01, # P <0.0001, ns, not significant, P > 0.05.

FIG. 7 is a graph showing that sulforaphane promotes the expression of the FSH receptor in granulosa cells in vivo in accordance with example 7 of the present invention. After the mice are injected into the abdominal cavity of sulforaphane, Western blot detects the protein level change of ovarian FSHR receptor and performs gray scale analysis. Results are expressed as standard deviation ± sem. P < 0.0001.

FIG. 8 is a graph of sulforaphane promoting ovarian follicular development and increasing follicular numbers in mice, in accordance with example 8 of the present invention. In the figure, A: and (3) detecting the ovarian morphology of the control group and the sulforaphane group after H & E staining of the mouse ovarian paraffin. B: after H & E, the number of ovarian preantral follicles (PA) and antral follicles (a) in mice was counted (n ═ 7). Results are expressed as standard deviation ± sem. P < 0.001.

FIG. 9 is a graph of increasing ovulation in mice promoted by sulforaphane according to example 9 of the present invention. In the figure, A: and counting the ovulation number of the mice in the control group and the sulforaphane group (n is 10). Results are expressed as mean ± sem. P < 0.001. B: the ovulation rate of the control group and the sulforaphane group was counted (n ═ 17).

FIG. 10 is a graphical representation of the overall effect of sulforaphane of the present invention in enhancing reproductive performance in mammals.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples were run under conventional experimental conditions, or as recommended by the manufacturer's instructions. The present invention will be described in further detail with reference to the following examples and accompanying drawings.

The important findings of the applicant group are that the sulforaphane obviously promotes the proliferation of follicular granular cells of pigs and mice and the synthesis of steroid hormones, and the sulforaphane promotes the ovarian weight gain, the follicular development and the ovulation of the mice. The weight of the ovary and the number of the antral follicles of the mouse are increased remarkably after 48 hours of in vivo administration of the sulforaphane, and particularly, the sulforaphane can be used for replacing FSH in the mouse superovulation program to induce the ovulation of the mouse. Meanwhile, the applicant also finds that the litter size of the daily ration of the weaned sows fed with the added sulforaphen for 3 days is obviously increased. Based on the above findings, applicants have invented techniques for using sulforaphane to improve reproductive performance in mammals. The technology for improving the reproductive performance of mammals by applying the sulforaphane provides a new application of the sulforaphane, greatly improves the application value of the sulforaphane, enlarges the application range of the sulforaphane, expands a new field of applying the sulforaphane, and has wide application prospect and commercial value.

Sulforaphane, used in the following examples, was purchased from Sigma (S4441) in vitro and from aladdin (S111997) in vivo.